Cell-Penetrating Peptide-Mediated Transformation of Large Plasmid

Apr 22, 2019 - In this technical note, we present a novel cell-penetrating peptide (CPP)-mediated large-sized plasmid DNA transformation system for ...
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Technical Note Cite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX

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Cell-Penetrating Peptide-Mediated Transformation of Large Plasmid DNA into Escherichia coli Md Monirul Islam,†,§ Masaki Odahara,† Takeshi Yoshizumi,†,∥ Kazusato Oikawa,† Mitsuhiro Kimura,†,∥ Masayuki Su’etsugu,‡ and Keiji Numata*,† †

Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan ‡ Department of Life Science, College of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo, 171-8501, Japan

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S Supporting Information *

ABSTRACT: The highly efficient genetic transformation of cells is essential for synthetic biology procedures, especially for the transformation of large gene clusters. In this technical note, we present a novel cell-penetrating peptide (CPP)-mediated large-sized plasmid DNA transformation system for Escherichia coli. A large plasmid (pMSR227, 205 kb) was complexed with cationic peptides containing a CPP motif and was successfully transformed into E. coli cells. The transformants containing the plasmid DNA exhibited expression of a reporter gene encoding a red fluorescent protein. The transformation efficiency was significantly higher than that obtained using the heat-shock method and was similar to that of electroporation. This technique can be used as a platform for the simple and highly efficient transformation of large DNA molecules under mild conditions without causing significant damage to DNA, accelerating synthetic biology investigations for the design of genetically engineered microorganisms for industrial purposes. KEYWORDS: cell-penetrating peptide, Escherichia coli, large-sized plasmid DNA, genetic material transformation

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electroporation can lead to high cell mortality, low transformation efficiency, and low throughput.16,17 Another common transformation method is heat-shock, which is widely used for bacteria due to its high throughput. However, a limitation of this technique is that it can only deliver smallsized plasmid DNA (pDNA) and chemically competent cells using calcium.18 In addition to electroporation and heat-shock methods, cellpenetrating peptide (CPP)-mediated transformation and gene delivery has been described by many groups using a wide variety of cells, such as bacteria, animal, and plant cells.19−25 In a previous study, the transfection of DNA with peptides was shown to prevent DNA damage and degradation.26 Thus, CPP-mediated DNA delivery is considered to be a suitable transformation/transfection technique for large DNA molecules. In this study, we report a CPP-mediated method for the transformation of large plasmid DNA into E. coli DH5α as a model Gram-negative bacterium. The large plasmid pMSR227 (205 kb) was used and encodes mCherry as a reporter gene.27 The CPP-mediated transformation of pMSR227 was successful and resulted in a similar transformation efficiency as electroporation but used an easier protocol than the heat-shock technique. This novel CPP-mediated method should aid in the

he use of genetic engineering and synthetic biology greatly contributes to the development of microbiome therapeutics,1 artificial photosynthesis,2 biomolecular manufacturing,3 in vivo diagnostics,4 and targeted cancer treatments.5 One of the primary steps in genetic engineering and synthetic biology is the delivery of genetic materials into cells efficiently and safely. However, there is no single technology that solves all of the existing problems for a wide variety of transformations. The delivery of genetic material through viral vectors, for example, is very efficient in both bacterial6 and mammalian cells,7 but this procedure is cell-type specific and has problems associated with immunogenicity.8 Cell squeezing is a new invention in the intracellular delivery of large molecules but is currently limited to mammalian cells.9,10 Conjugation is widely used for the delivery of DNA into Escherichia coli,11 mycobacteria,12 and Bacillus but has problems with cell-type specificity and is not suitable for many other bacterial species.13 Since the 1980s, electroporation (EP) has been widely used to introduce genetic material into both mammalian and bacterial cells. Additionally, EP can be used to deliver very large-sized plasmids, such as bacterial artificial chromosomes (BACs, typically 150−350 kb).14 This method is strongly dependent on the electric field strength experienced by the cells, as field strengths that are too high cause irreversible electroporation, resulting in cell lysis and death.15 Electroporation is not species specific, but without optimization, © XXXX American Chemical Society

Received: February 7, 2019

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DOI: 10.1021/acssynbio.9b00055 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Technical Note

ACS Synthetic Biology manipulation of large DNA molecules in the field of synthetic biology.

heat-shock method. The extracted pDNA from the transformants exhibited the appropriate DNA size by pulsed field gel electrophoresis (Figure 1b). Furthermore, the extracted pDNA was digested with the restriction endonuclease XhoI and produced a digestion pattern that was identical to that obtained for pMSR227 in a previous study (DNA fragment sizes of 52, 43, 38, 29, 14.4, 10.4, 7.5, 6.4, and 4.4 kb).27 Transformations using 1 and 2 μg of pDNA showed the highest transformation efficiency at an N/P of 0.1 (Figure S4). We also normalized the transformation efficiencies by the competency of E. coli DH5a competent cells, because of the significant difference in competency between chemical and electrocompetent cells (Figure S5). With respect to the transformation efficiency normalized by their competency (CFUs obtained per number of competent cells), the complexes prepared at an N/P of 0.1 showed the highest efficiency, which was almost 3.5-fold higher than that obtained by electroporation. Based on these results, 1 μg of the pDNA complex prepared at an N/P of 0.1 was identified as the optimal condition for the transformation of pMSR227 into E. coli DH5α cells using (KH)9-BP100. To confirm the transformation, the expression of a reporter gene, mCherry, was evaluated by confocal laser scanning microscopy (CLSM). The E. coli cells transformed with the pMSR227 and (KH)9-BP100 complexes exhibited notable fluorescent mCherry signals (Figure 2 and Figure S6). Thus, the CPP-mediated transformation of E. coli cells with large pDNA was successful and exhibited a relatively high efficiency.

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RESULTS The peptide (KH)9-BP100 (amino acid sequence, KHKHKHKHKHKHKHKHKHKKLFKKILKYL-NH2; theoretical pI/ Mw, 10.81/3809.71 Da), which was designed as a fusion peptide of the CPP BP100 and the polycation (KH)9,28 was used to transform the large plasmid pMSR227.27 Ionic complexes of (KH)9-BP100 and pDNA were prepared at different N/P ratios ranging from 0.01 to 5, with the N/P ratio referring to the ratio of the number of amine groups from the peptide to the number of phosphate groups from pDNA. The hydrodynamic diameters, zeta-potentials, and electrical stability of the complexes were evaluated, which confirmed the expected complex formation (Figures S1 and S2, Table S1). The transformation efficiency of the pMSR227 and (KH)9BP100 complexes was evaluated by determining the number of colony forming units (CFUs) obtained per microgram of pDNA (Figure 1a and Figure S3). For the 205-kb pDNA transformation assay, the complex prepared at an N/P of 0.1 showed the highest transformation efficiency, which was similar to that obtained via electroporation and was approximately 5-fold higher than that obtained using the

Figure 2. CLSM images of mCherry expression in E. coli DH5α cells. Overlay of mCherry and DIC images: (a) E. coli DH5α transformants harboring pMSR227 encoding mCherry and (b) wild-type E. coli DH5α cells as a negative control.

CPP-mediated transformation is an ideal method for the genetic transformation of microbes due to its high transformation efficiency and high throughput without any special equipment. In addition, the cationic peptide can condense and protect nucleic acids from external biological and physical attacks.26 In this study, the ability to transform very large pDNA (up to 205 kb) into E. coli cells is demonstrated via cationic CPP-mediated transformation. Further optimization (e.g., the use of another type of CPP, peptide sequences, and conditions) could potentially increase the transformation efficiency for wild-type E. coli DH5α and other bacterial strains. Ultimately, the concepts presented here will result in a flexible transformation platform customizable to a wide array of microbial cells for synthetic biology.

Figure 1. CPP-mediated transformation of pMSR227 into E. coli DH5α cells. (a) Transformation efficiency (CFU/μg of pDNA) from electroporation (E), heat-shock (0H), and CPP-mediated transformation at different N/P ratios. Each data point represents the average of the eight separate trials (n = 8). Error bars show the standard deviation. *Significant difference by t test (p < 0.01). (b) Pulsed field gel electrophoresis of intact pMSR227 (205 kb), which was extracted from the CPP-mediated transformants. “λ” denotes λDNA marker and “PC” denotes intact pMSR227 (positive control). (c) Pulsed field gel electrophoresis of the extracted pDNA after digestion by the restriction endonuclease XhoI.

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EXPERIMENTAL PROCEDURES CPP-Mediated Transformation for E. coli. Detailed protocols for the preparation of (KH)9-BP100, pMSR227 (205 kb), and E. coli DH5α competent cells for heat shock (chemical competent cells) and electroporation are provided B

DOI: 10.1021/acssynbio.9b00055 ACS Synth. Biol. XXXX, XXX, XXX−XXX

ACS Synthetic Biology in the Supporting Information. The protocols used for the characterization of peptide−pDNA complexes and CLSM for the detection of mCherry expression are also summarized in the Supporting Information, as are the details of the statistical analyses performed. (KH)9-BP100 and pMSR227 complexes were prepared by adding of 2.5 mg/mL pMSR227 to increasing volumes of CPP at various N/P ratios (0.05, 0.1, 0.5, 1, 2 and 5) and autoclaved Milli-Q water to obtain a final volume of 100 μL. The solutions were thoroughly mixed and allowed to stabilize for 30 min at 25 °C. The solutions containing the complexes were mixed with 200 μL of E. coli DH5α cells by pipetting and allowed to transform for 30 min at 25 °C. Subsequently, 1 mL of LB broth was added to the tubes, which were then incubated at 37 °C for 1 h. Next, the tubes were centrifuged for 1 min at 8000 rpm, and approximately 100 μL was plated onto agar-solidified LB broth plates. The transformation efficiency was calculated by following eq 1.

number of colonies on plate (df) × 1000 ng/μg amount of DNA plated (ng)

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(1)

(2)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.9b00055.

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ACKNOWLEDGMENTS

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REFERENCES

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After an overnight incubation, images of the agar plates were taken with a Nikon digital camera (Nikon, Tokyo, Japan). The number of colony forming units (CFUs) was determined using ImageJ (National Institutes of Health, Bethesda, MD)29 and eq 1. Transformation efficiency was defined as the number of CFUs on kanamycin-containing LB agar plates obtained per microgram of pDNA. Standardized transformation efficiency (%) was calculated using eq 2. standardized transformation efficiency (%) [CFU] /μg DNA = × 100 total number of cells plated (ng)

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This work was supported by a JST ERATO Grant (Grant JPMJER1602) (K.N.) and ImPACT (T.Y.).

transformation efficiency by colony forming unit (CFU) =

Technical Note

Full experimental procedures, results of the complex characterization, and additional characterization data and spectra (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Keiji Numata: 0000-0003-2199-7420 Present Addresses §

M.M.I.: Department of Bioscience and Biotechnology, The University of Suwon, Hwaseong City, Gyeonggi-Do 18323, Republic of Korea. ∥ T.Y. and M.K.: Takasaki University of Health and Welfare, 37-1 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan. Notes

The authors declare no competing financial interest. C

DOI: 10.1021/acssynbio.9b00055 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Technical Note

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DOI: 10.1021/acssynbio.9b00055 ACS Synth. Biol. XXXX, XXX, XXX−XXX